1. Technical Field
The present method and testing medium relate to the detection of pathogenic staphylococci, such as Staphylococcus aureus (“S. aureus”) in a biological, environmental, or food sample, and more particularly to those methods and testing media utilizing reacting factors with which the targeted microbe(s) can produce one or both of a detectable signal in a hydrated mixture of the medium and sample being tested.
2. Background Information
Pathogenic staphylococci (“P. staphylococci”) can be a virulent pathogen of animals and humans. Moreover, it can cause severe food poisoning by the production of a toxin. Diseases caused by P. staphylococci cover a very wide clinical spectrum, from simple skin infections to life threatening infections of the bones, heart, and organs. Of particular concern is the recognition that P. staphylococci infection is common after surgery. It is also associated with intravenous tubing and other implants.
The bacterium P. staphylococci may be transmitted between healthy individuals by skin to skin contact, or from a commonly shared item or a surface (e.g., tanning beds, gym equipment, food handling equipment, etc.) where the transfer may be made to a subsequent person who uses the shared item or touches the surface. Of great medical concern is the recognition that healthy people entering hospitals may “carry” P. staphylococci bacteria (e.g., on their skin, or in their noses, etc.) without any signs or symptoms that they do so. In the presence of favorable conditions (often found in, but not limited to, hospitals), the P. staphylococci bacteria can activate and cause serious infection. In addition, P. staphylococci bacteria can also be a source of food poisoning, often caused by a food handler contaminating the food product (e.g., meat, poultry, eggs, salads containing mayonnaise, bakery products, dairy products, etc.).
P. staphylococci bacteria is often, but not always, classified based on an individual clone's susceptibility to the class of antibiotics such as methicillin; e.g., methicillin susceptible S. aureus (MSSA), and methicillin resistant S. aureus (MRSA). Methicillin is a narrow spectrum beta-lactam antibiotic of the penicillin class. Until only a few years ago, MRSA was most commonly found in hospitals. Now, it is frequently also present in the noses, skin, etc. of people in the non-hospital community. Moreover, these MRSA bacteria are increasingly causing serious infections in the community. MRSA is particularly serious because only very few antibiotics (e.g., vancomycin) have been shown to be uniformly effective against MRSA. Vancomycin-resistant Staphylococcus aureus is another class of P. staphylococci bacteria. Vancomycin-resistant Staphylococcus aureus refers to strains of S. aureus that have become resistant to the glycopeptide antibiotic vancomycin. With the increase of staphylococcal resistance to methicillin, vancomycin (or another glycopeptide antibiotic; e.g., teicoplanin) is often used to treat infections with methicillin-resistant S. aureus (MRSA). Three classes of vancomycin-resistant S. aureus have emerged that differ in vancomycin susceptibilities: vancomycin-intermediate S. aureus (VISA), heterogenous vancomycin-intermediate S. aureus (hVISA), and high-level vancomycin-resistant S. aureus (VRSA). Other classes of P. staphylococci bacteria are associated with antibiotics including: linezolid, clindamycin, erythromycin, tetracycline, and sulfa-trimethoprim.
The Center for Disease Control and Prevention actively surveys for the development of MRSA. In 2000, the Society for Healthcare Epidemiology of America guidelines recommended contact isolation for patients with MRSA. In addition to the morbidity and mortality caused by MRSA, it has been estimated that each case of infection costs at least $23,000. Accordingly, many hospitals and nursing homes proactively sample patients for MRSA. See Clany, M., Active Screening in High-Risk units is an effective and cost-avoidant method to reduce the rate of methicillin-resistant Staphylococcus aureus infection in the hospital, Infection Control and Hospital Epidemiology 27: 1009-1017, 2006.
Meyer et al. (U.S. Pat. No. 4,035,238) describes the use of a broth for the detection of S. aureus that utilizes mannitol as a source of carbon and DNA meth. Neither of these chemicals are coagulase reactive substrates.
Rambach (U.S. Pat. No. 6,548,268) employs at least two chromogenic agents in an agar medium: 5-bromo-6-chloro-indoxyl-phosphate; and 5-bromo-4-chloro-3-indoxyl glucose in the presence of desferoxamine. An individual colony hydrolyzing these substrates will produce colors that will mix with each other and not be independent of one another.
A large number of classical culturing procedures are utilized to detect MSSA and MRSA from samples collected from humans, animals, food, etc. The culturing procedures share a basic medium (e.g., a trehalose-mannitolphosphatase agar) with chemical inhibitors such as 6-8% sodium chloride, potassium tellurite, and a variety of antibiotics; See Stevens, D. L. and Jones, C., “Use of trehalose-mannitol-phosphatase agar to differentiate Staphylococcus epidermidis and Staphylococcus saprophyticus from other coagulase-negative staphylococci”, J. of Clin. Microbiology 20:977-980, 1984. The use of mannitol as a carbon source and salt as a selective agent into an agar known as mannitol-salt agar has been commonly used in clinical laboratories; See Baird, R. M. and Lee, W. H., “Media used in the detection and enumeration of Staphylococcus aureus., Int. J. Food Microbiology. 26:209-211, 1995. Within the prior art of culturing, it is a generally accepted procedure to perform coagulase tests utilizing samples of S. aureus that are isolated in a pure culture as a required test to achieve sufficient specificity.
The procedure “S. aureus 10”, available from bioMérieux, Inc., Durham, N.C., USA, uses an alpha-glucosidase substrate in agar to detect S. aureus. A single substrate is utilized; See Perry, J. D. et al., “Evaluation of S. aureus 10, a new chromogenic agar medium for detection of Staphylococcus aureus”, J. Clin. Microbiology 41: 5695-5698, 2003. A variant of this medium, which contains added antibiotics and sodium chloride, is designed to detect MRSA; See Perry et al., “Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus”, J. of Clin. Micro. 42: 4519-4523, 2004.
Selepak and Witebsky disclose a study evaluating the inoculum size and lot-to-lot variability of the tube coagulase test for S. aureus. Specimens were collected and isolates were generated from the bacterial colonies on agar plates. Tubes containing anticoagulated rabbit coagulase plasma were inoculated with a part of, or more than one, staphylococcal colony from the isolates. The tubes were incubated and examined for the presence of clot. According to Selepak and Witebsky “with some isolates and some lots of coagulase plasma, even a single colony [from the isolate] may not provide enough inoculum for a positive coagulase test”. Furthermore, Selepak and Witebsky state that “[e]xpressed more quantitatively, at least 108 organisms per ml should be used whenever possible for each coagulase tube test. Our data further suggests that S. aureus does not grow in coagulase plasma; therefore, the incubation of coagulase plasma for 18 to 24 h does not compensate for the use of small inoculum.” Thus, Selepak and Witebsky indicate that it is impractical, if not impossible, to detect the presence or absence of S. aureus in first generation biological specimen samples using a direct coagulase test. See Selepak, S. T et al, “Inoculum Size and Lot-to-Lot Variation as Significant Variables in the Tube Coagulase Test for Staphylococcus aureus”, Journal of Clin. Microbiology, November 1985, p. 835-837]
Orth and Anderson disclose the use of a mannitol coagulase agar for the detection of S. aureus. The disclosure technique detects S. aureus by a fibrin deposition around the colony, and does not disclose a technique wherein detection is predicated on the formation of a gel. In addition, the technique is used to detect all stains of S. aureus indiscriminately. See, Orth, D. S. and Anderson, A. W., “Polymyxin—Coagulase—Mannitol—Agar”, Applied Microbiology, January 1970, pp. 73-75.
It would, therefore, be desirable to provide a test mixture and a method that can rapidly detect a targeted antibiotic resistant strain of P. staphylococci directly from a first generation sample, one that does not require a skilled technician to perform the method, one that does not require the use of isolates developed from the specimen sample (i.e., one that can be performed on a “first generation” specimen sample) but one that can be used on such isolates, one that does not require a large concentration of S. aureus organisms to be accurate, and one that is stable at room temperatures for an extended time period.